生物化学资料：1-DNA Replication.pptVIP

TOPICS OUTLINE 1. Prokaryotic DNA Biosynthesis 2. Eukaryotic DNA Biosynthesis 3. Reverse transcription 4. Organization of DNA (chromosome) 5. DNA roles 6. DNA damage and repair semi-conservative replication high fidelity bidirectional replication semi-discontinuous replication DNA polymerase: DNA-dependent DNA polymerase, DNA-pol The DNA Polymerase Family A total of 5 different DNA Pols have been reported in E. coli DNA Pol I: functions in repair and replication DNA Pol II: functions in DNA repair (proven in 1999) DNA Pol III: principal DNA replication enzyme DNA Pol IV: functions in DNA repair (discovered in 1999) DNA Pol V: functions in DNA repair (discovered in 1999) Why the exonuclease activities? The 3-5 exonuclease activity serves a proofreading function It removes incorrectly matched bases, so that the polymerase can try again. In 1957, Arthur Kornberg demonstrated the existence of a DNA polymerase - DNA polymerase I DNA Polymerase I has THREE different enzymatic activities in a single polypeptide: a 5’ to 3’ DNA polymerizing activity a 3’ to 5’ exonuclease activity a 5’ to 3’ exonuclease activity Is DNA Polymerase I the principal replication enzyme?? In 1969 John Cairns and Paula deLucia isolated a mutant bacterial strain with only 1% DNA Pol I activity (polA) Conclusion: DNA Pol I is NOT the principal replication enzyme in E. coli Other clues…. - DNA-Pol I is too slow (600 dNTPs added/minute – would take 100 hrs to replicate genome) Conclusion: There must be additional DNA polymerases. Biochemists purified them from the polA mutant So if it’s not the chief replication enzyme then what does DNA-Pol I do? - functions in multiple processes that require only short lengths of DNA synthesis - has a major role in DNA repair (Cairns- deLucia mutant was UV-sensitive) - its role in DNA replication is to remove primers and fill in the gaps left behind - for this it needs the nick-translation activity DNA Polymerase III